Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

Background  

Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca²⁺-activated K⁺ channel (K_Ca) of big conductance (BK_Ca), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca²⁺ levels. In this study, we aimed at characterizing 1. expression and functions of K_Ca channels (including BK_Ca beta-subunits), and 2. biophysical properties of BK_Ca channels.     

cAMP controls cytosolic Ca<Superscript>2+</Superscript> levels in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) that is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca²⁺]_i.     

Relationship between Extra- and Intracellular Calcium in Distal Segments of the Renal Tubule. Role of the Ca2+ Receptor RaKCaR

The effect of extracellular calcium ([Ca²⁺] _e ) on cytosolic calcium ([Ca²⁺] _i ) was investigated in thick ascending limbs and collecting ducts from the rat kidney, using the fluorescent dye fura-2. In cortical collecting ducts, basolateral but not apical changes in [Ca²⁺] _e were associated with parallel changes in [Ca²⁺] _i . Basal [Ca²⁺] _i was hardly modified by nifedipine and verapamil but was decreased by 60% by basolateral La³⁺. Increasing peritubular [Ca²⁺] _e triggered Ca²⁺ release from intracellular stores. This effect was not reproduced by agonists of the renal Ca²⁺-receptor RaKCaR, e.g., Ba²⁺, Mg²⁺, Gd³⁺, and neomycin, but was reproduced by Ni²⁺. Ni²⁺-induced mobilization of intracellular Ca²⁺ was larger in the inner medullary collecting duct, a segment which poorly responds to increasing [Ca²⁺] _e . In the cortical thick ascending limb, removing basolateral Ca²⁺ hardly altered [Ca²⁺] _i but increasing [Ca²⁺] _e or adding Ba²⁺, Mg²⁺, Gd³⁺ and neomycin released intracellular calcium. These data demonstrate that (1) basolateral influx of calcium occurs in cortical collecting ducts, under basal conditions; (2) this influx occurs through nonvoltage gated channels, permeable to Ba²⁺, insensitive to verapamil and nifedipine, and blocked by La³⁺; (3) increasing [Ca²⁺] _e stimulates the influx and triggers intracellular calcium release, independently of the phospholipase C-coupled receptor RaKCaR; (4) RaKCaR is functionally expressed in thick ascending limbs; (5) another membrane receptor, sensitive to Ni²⁺ but not to Ca²⁺ is present in the collecting duct. Received: 12 July 1996/Revised: 28 October 1996     

Oxidant Injury in PC12 Cells—A Possible Model of Calcium "Dysregulation" in Aging: I. Selectivity of Protection Against Oxidative Stress

Abstract: Previous research has suggested that the initial effects of cellular free radical neurotoxic insult involve large increases in intracellular Ca²⁺. However, the exact role of oxidative stress on the various parameters involved in these increases has not been specified. The present experiments were performed to examine these parameters in PC12 cells exposed to 5, 25, or 300 µM H₂O₂ for 30 min in growth medium alone or containing either nifedipine (L-type Ca²⁺ antagonist), conotoxin (N-type antagonist), Trolox (vitamin E analogue), or α-phenyl-n-tert-butylnitrone (nitrone trapping agent; PBN). The concentrations of H₂O₂ were chosen by examining the degree of cell killing induced by exposure to graded concentrations of H₂O₂. The 5 and 25 µM concentrations of H₂O₂ produced no significant cell killing at either 30 min or 24 h after treatment, whereas the 300 µM concentration produced a moderate degree of cell killing that did not increase between the two times. Fluorescent imaging was used to visualize intracellular Ca²⁺ changes in fura-2-loaded cells. Baseline (pre-30 mM KCI) Ca²⁺ levels were increased significantly by H₂O₂ treatment (e.g., 300 µM, 200%), but the rise in the level of free intracellular Ca²⁺ after KCI stimulation (i.e., peak) was decreased (e.g., 300 µM, 50%) and the cell's ability to sequester or extrude the excess Ca²⁺ (i.e., Ca²⁺ recovery time) after depolarization was decreased significantly. All compounds prevented baseline Ca²⁺ increases and, with the exception of conotoxin, antagonized the peak decreases in Ca²⁺. It is interesting that after 300 µM H₂O₂ exposure, only Trolox was partially effective in preventing these deficits in recovery. Conotoxin increased the decrement recovery in the absence of H₂O₂. However, in cells exposed to 5 or 25 µM H₂O₂, conotoxin as well as the other agents were effective in preventing the deficits in recovery.     

Kinetic analysis of excitation-contraction coupling

Recent studies of isolated muscle membrane have enabled induction and monitoring of rapid Ca²⁺ release from sarcoplasmic reticulum (SR)⁵ in vitro by a variety of methods. On the other hand, various proteins that may be directly or indirectly involved in the Ca²⁺ release mechanism have begun to be unveiled. In this mini-review, we attempt to deduce the molecular mechanism by which Ca²⁺ release is induced, regulated, and performed, by combining the updated information of the Ca²⁺ release kinetics with the accumulated knowledge about the key molecular components.Abbreviations used: AMP-PCP, adenosine 5-(, -methylenetriphosphate); C_1/2, concentration a half-maximal activation or inhibition; Con-A, concanavalin A; DACM,N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide; DCCD, dicyclohexylcarbodiimide; SR, sarcoplasmic reticulum; DHP, dihydropyridine; E-C, excitation-contraction; EP, phosphorylated intermediate of the enzyme; IP₃, inositol 1,4,5-trisphosphate; JFM, junctional face membrane;M _r, molecular weight; T-tubule, transverse-tubular system.     

Ca<Superscript>2+</Superscript> regulation in the absence of the <Emphasis Type="Italic">iplA</Emphasis> gene product in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). The [Ca²⁺]_i-change is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-entry. The significance of the [Ca²⁺]_i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca²⁺-buffers in the cytosol indicates that a [Ca²⁺]_i-increase is required for chemotaxis. Yet, the iplA ^- mutant disrupted in a gene bearing similarity to IP₃-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca²⁺]_i-transient which favours the view that [Ca²⁺]_i-changes are insignificant for chemotaxis.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.     

Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins

Background  

A wide range of stimuli evoke rapid and transient increases in [Ca²⁺]_cyt in plant cells which are transmitted by protein sensors that contain EF-hand motifs. Here, a group of Oryza sativa L. genes encoding calmodulin (CaM) and CaM-like (CML) proteins that do not possess functional domains other than the Ca²⁺-binding EF-hand motifs was analyzed.     

Excitability of Paramecium and the significance of negative surface charges

Summary On the basis of a model presented in a previous paper (Hook and Hildebrand, 1979) the influence of external cation concentrations [K⁺]₀, [Ca²⁺]₀ and of membrane voltage V_m (i.e. the actual potential difference between the two membrane faces) on the locomotor behavior of Paramecium is theoretically analyzed. In an extended model system we discuss the negative feedback of intraciliary calcium [Ca²⁺]_i on the excitability of the ciliary membrane. While a fast blocking of Ca channels is mediated by increased [Ca²⁺]_i and accounts for the short duration of action potentials, a slow [Ca²⁺ ]_i-dependent denaturation of channel molecules is assumed to determine excitability changes of Paramecium on a long time scale.It is emphasized that the duration of long-lasting ciliary reversal which reflects the excitability is not a direct function of the cation ratio J_u [K⁺]₀/[Ca²⁺] ₀ ^1/2 but rather of the membrane potential V_m.Introduction of negative surface charges can well explain why for a series of different [K⁺]₀, [Ca²⁺]₀ but constant J_a value the excitability is unchanged despite corresponding shifts in measured membrane potentials.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

A new short-term toxicity assay using <Emphasis Type="Italic">Aspergillus awamori</Emphasis> with recombinant aequorin gene

Background  

Most currently available short-term toxicity assays are based on bacterial cells. Therefore there is a need for novel eukaryotic microbial bioassays that will be relevant to higher eukaryotes such as animals and plants. Ca²⁺ is a universal intracellular signalling molecule found in all organisms from prokaryotes to highly specialized animal cells. In fungi calcium has been demonstrated to be involved in control of many important processes. The recombinant aequorin gene from the jellyfish Aequorea victoria responsible for the expression of the Ca²⁺-sensitive aequorin photoprotein has been cloned in the filamentous fungus Aspergillus awamori. This has allowed real life monitoring of [Ca²⁺]_c changes in living fungal cells. When subjected to different physico-chemical stimuli fungal cells respond by transiently changing the concentration of free Ca²⁺ in the cytosol ([Ca²⁺]_c) and the pattern of these changes (Ca²⁺ signature) is specific to each particular stimulus. Therefore it was interesting to investigate whether different environmental toxicants would be able to affect the pattern of [Ca²⁺]_c changes in a reproducible and dose dependant manner.     

Disruption of actin filaments induces mitochondrial Ca<Superscript>2+</Superscript> release to the cytoplasm and [Ca<Superscript>2+</Superscript>]<Subscript>c</Subscript> changes in <Emphasis Type="Italic">Arabidopsis</Emphasis>root hairs

Background  

Mitochondria are dynamic organelles that move along actin filaments, and serve as calcium stores in plant cells. The positioning and dynamics of mitochondria depend on membrane-cytoskeleton interactions, but it is not clear whether microfilament cytoskeleton has a direct effect on mitochondrial function and Ca²⁺ storage. Therefore, we designed a series of experiments to clarify the effects of actin filaments on mitochondrial Ca²⁺ storage, cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c), and the interaction between mitochondrial Ca²⁺ and cytoplasmic Ca²⁺ in Arabidopsis root hairs.     

Involvement of ATP,increase of intracellular calcium and the early expression of <Emphasis Type="Italic">c-fos</Emphasis> in the repair of rat fetal articular cartilage

To compare the potential of adult and fetal animals to repair articular cartilage, we investigated the early process after creating superficial defects in the femoral knee cartilage in rat models. In fetuses at 19 days of gestation, both chondrocytes and the extracellular matrix responded notably by 48 h after artificial injury. Staining patterns with safranin O revealed that, by 1 h after injury, some components of the extracellular matrix around the wound were modified, and the change spread from the limited region to the entire knee cartilage within 24 h. The chondrocytes in the area surrounding the wound transiently expressed increased level of c-fos from 1 h to 6 h. The wound remained 1 day after birth, i.e., 72 h after injury, but was completely repaired 10 days after birth. In contrast, neither visible responses nor transient c-fos expression was observed in 12-week-old adult articular cartilage 48 h after injury. We also examined the relationships between the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the induction of c-fos expression in the cartilage. Applications of ATP or Ca²⁺ ionophore A23187, both of which increase [Ca²⁺]_i, induced immediate expression of c-fos in primary cultured chondrocytes: 1 M ATP elicited an increase of [Ca²⁺]_i in chondrocytes in fetal cartilage slices, but 1 mM was required in adult cartilage slices. Our findings show the presence of a signaling pathway that is apparently active in the repair of fetal but not adult articular cartilage and that involves the intercellular transfer of ATP, increase of [Ca²⁺]_i, and expression of c-fos in cartilage.This study was supported in part by Health Sciences Research Grants for Research on Human Genome, Tissue Engineering and Food Biotechnology to M.O. from the Japanese Ministry of Health, Labor and Welfare     

Calcium and tip growth inNeurospora crassa

Summary We examined the ionic regulation of tip growth inNeurospora crassa by a combination of electrophysiology and confocal microscopy. To determine if transmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from –200 to +50 mV. In this voltage range, transmembrane ionic fluxes would either reverse (e.g., K⁺) or change dramatically (e.g., Ca²⁺ influx) but had no effect on hyphal growth rates. Therefore, ionic fluxes (including Ca²⁺ influx) may not be required for tip growth. However, intracellular Ca²⁺ may still play an obligatory role in tip growth. To assess this possibility, we first increased cytosolic Ca²⁺ directly by ionophoresis. Elevated Ca²⁺ induced subapical branch initiation, often multiple tips. At hyphal tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pronounced tip-high Ca²⁺ gradient within 10 m of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca²⁺ chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate consistently inhibited growth concomitantly with a depletion of intracellular Ca²⁺ and dissipation of the tip-high gradient. We conclude that Ca²⁺ plays a regulatory role in tip initiation and the maintenance of tip growth. Because plasma membrane ionic fluxes do not play a role in tip growth, we suggest that the tip-high Ca²⁺ gradient is generated from intracellular Ca²⁺ stores in the ascomyceteN. crassa.Abbreviations BAPTA 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate - [Ca²⁺]_i intracellular Ca²⁺ concentration - fluo-3 2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl-4-methyl-2,2-(ethylenedioxy)dianiline-N,N,N,N-tetraacetic acid     

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation

Background

All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP₃Rs)-binding (CIRB) site involved in the regulation of TRPC channel function.

Objectives

To assess the basis of CaM/IP₃Rs binding to the CIRB site of TRPC6 and its role in platelet physiology.

Methods

Protein association was detected by co-immunoprecipitation and Western blotting, Ca²⁺ mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry.

Results

Co-immunoprecipitation of TRPC6 with CaM or the IP₃Rs at different cytosolic free Ca²⁺ concentrations ([Ca²⁺]_c) indicates that the association between these proteins is finely regulated by cytosolic Ca²⁺ via association of CaM and displacement of the IP₃Rs at high [Ca²⁺]_c. Thrombin-stimulated association of TRPC6 with CaM or the IP₃Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca²⁺-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP₃Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca²⁺ entry in the presence of low or high, respectively, extracellular Ca²⁺ concentrations, as well as altered thrombin-evoked platelet aggregation.

Conclusions

Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca²⁺ entry and aggregation through its interaction with CaM and IP₃Rs.     

Acid-sensing ion channel 3 decreases phosphorylation of extracellular signal-regulated kinases and induces synoviocyte cell death by increasing intracellular calcium

Introduction

Acid-sensing ion channel 3 (ASIC3) is expressed in synoviocytes, activated by decreases in pH, and reduces inflammation in animal models of inflammatory arthritis. The purpose of the current study was to characterize potential mechanisms underlying the control of inflammation by ASIC3 in fibroblast-like synoviocytes (FLS).

Methods

Experiments were performed in cultured FLS from wild-type (WT) and ASIC3-/- mice, ASIC1-/- mice, and people with rheumatoid arthritis. We assessed the effects of acidic pH with and without interleukin-1β on FLS and the role of ASICs in modulating intracellular calcium [Ca²⁺]_i, mitogen activated kinase (MAP kinase) expression, and cell death. [Ca²⁺]_i was assessed by fluorescent calcium imaging, MAP kinases were measured by Western Blots; ASIC, cytokine and protease mRNA expression were measured by quantitative PCR and cell death was measured with a LIVE/DEAD assay.

Results

Acidic pH increased [Ca²⁺]_i and decreased p-ERK expression in WT FLS; these effects were significantly smaller in ASIC3-/- FLS and were prevented by blockade of [Ca²⁺]_i. Blockade of protein phosphatase 2A (PP2A) prevented the pH-induced decreases in p-ERK. In WT FLS, IL-1β increases ASIC3 mRNA, and when combined with acidic pH enhances [Ca²⁺]_i, p-ERK, IL-6 and metalloprotienase mRNA, and cell death. Inhibitors of [Ca²⁺]_i and ERK prevented cell death induced by pH 6.0 in combination with IL-1β in WT FLS.

Conclusions

Decreased pH activates ASIC3 resulting in increased [Ca²⁺]_i, and decreased p-ERK. Under inflammatory conditions, acidic pH results in enhanced [Ca²⁺]_i and phosphorylation of extracellular signal-regulated kinase that leads to cell death. Thus, activation of ASIC3 on FLS by acidic pH from an inflamed joint could limit synovial proliferation resulting in reduced accumulation of inflammatory mediators and subsequent joint damage.     

Modeling CICR in rat ventricular myocytes: voltage clamp studies

Background  

The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect Ca ²⁺ loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca ²⁺ release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic Ca ²⁺ concentration ([Ca ²⁺] _myo ).     

A comparison of the effects of Ca2+ and gibberellic acid on enzyme synthesis and secretion in barley aleurone

The effects of the addition and withdrawal of gibberellic acid (GA₃) and Ca²⁺ on enzyme synthesis and secretion by barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. Incubation of layers in GA₃ plus Ca²⁺ affects the total amount of secreted α-amylase (EC 3.2.1.1) and acid phosphatase (EC 3.1.3.2) by promoting the appearance of different isoenzymic forms of these enzymes. The release of α-amylase isoenzymes 1–4 in response to GA₃ plus Ca²⁺ has a lag of 6 h. When layers are incubated in GA₃ alone for 6 h prior to the addition of Ca²⁺, isoenzymes 1–4 appear in the medium after only 30 min. When the addition of Ca²⁺ to layers pretreated in GA₃ is delayed beyond 12 h, its effectiveness in stimulating the synthesis and release of isoenzymes 3 and 4 is diminished. After 35 h of preincubation in GA₃, addition of Ca²⁺ will not stimulate synthesis of α-amylase isoenzymes 3 and 4. Aleurone layers preincubated for 6 h in GA₃ will respond to Ca²⁺ when the GA₃ is withdrawn from the incubation medium by producing α-amylase isoenzymes 1–4. The converse is not the case, however, since layers preincubated in Ca²⁺ for 6 h will not produce all isoenzymes of α-amylase when subsequently incubated in GA₃. The Ca²⁺-stimulated release of α-amylase from GA₃ pre-treated layers is dependent on the time of incubation in Ca²⁺ and the concentration of the ion. The response to Ca²⁺ is temperature-dependent, and other divalent cations such as Mg²⁺ cannot substitute for Ca²⁺. We conclude that Ca²⁺ influences α-amylase release by influencing events at the biochemical level.     

Production,purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l⁻¹ in an optimized medium containing 20 g of malt extract l⁻¹, 2 g of yeast extract l⁻¹, 1.5 mM CuSO₄. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten constant (K _m ) of the laccase was 18 μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70°C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, while inhibited by 4.0 mM of Co²⁺, Al³⁺, Cu²⁺, and Fe²⁺, showing different profiles of metal ion effects.     

Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei

Crustaceans present a very interesting model system to study the process of calcification and calcium (Ca²⁺) transport because of molting-related events and the deposition of CaCO₃ in the new exoskeleton. Dilocarcinus pagei, a freshwater crab endemic to Brazil, was studied to understand Ca²⁺ transport in whole gill cells using a fluorescent probe. Cells were dissociated, all of the gill cell types were loaded with fluo-3 and intracellular Ca²⁺ change was monitored by adding Ca as CaCl₂ (0, 0.1, 0.25, 0.50, 1.0 and 5 mM), with a series of different inhibitors. For control gill cells, Ca²⁺ transport followed Michaelis–Menten kinetics with K _m = 0.42 ± 0.04 mM and V _max = 0.50 ± 0.02 μM (Ca²⁺ change × initial intracellular Ca⁻¹ × 180 s⁻¹; N = 14, r ² = 0.99). Verapamil (a Ca²⁺ channel inhibitor) and amiloride (a Na⁺/Ca²⁺ exchanger [NCX] inhibitor) completely reduced intracellular Ca²⁺ transport, while nifedipine, another Ca²⁺ channel inhibitor, did not. Vanadate, a plasma membrane Ca²⁺-ATPase inhibitor (PMCA), increased intracellular Ca²⁺ in gill cells through a decrease in the efflux of Ca²⁺. Ouabain increased intracellular Ca²⁺, similar to the effect of KB-R, a specific NCX inhibitor for Ca²⁺ in the influx mode. Alterations in extracellular [Na] in the saline did not affect intracellular Ca²⁺ transport. Caffeine, responsible for inducing Ca release from sarcoplasmic reticulum in vertebrate muscle, increased intracellular Ca²⁺ compared to control, suggesting an effect of this inhibitor in gill epithelial cells of Dilocarcinus pagei, probably through release of intracellular stores. We also demonstrate here that intracellular Ca²⁺ in gill cells of Dilocarcinus pagei was kept relatively constant in face of an extracellular Ca concentration of 50-fold, suggesting that crustaceans are able to display Ca²⁺ homeostasis through various Ca²⁺ intracellular sequestration mechanisms and/or plasma membrane Ca²⁺ influx and outflux that are highly regulatory. In summary, studies using whole gill cells are an interesting approach for working with real regulatory Ca²⁺ mechanisms in intact cells under physiological Ca levels (mM range), compared to earlier work using isolated vesicles of various epithelial cells.